Stimuli-responsive polymer utilizing keto-enol tautomerization and stimuli-responsive separating material and chemical-releasing capsule comprising the same

ABSTRACT

A stimuli-responsive polymer derivative utilizing keto-enol tautomerization. Also disclosed are a simple process for producing an N-acyl(meth)acrylamide derivative which can be used as a monomer for the stimuli-responsive polymer, a process for the production of an intermediate thereof, and an intermediate thus produced.

This application is a divisional application of Ser. No. 10/178,474,filed Jun. 25, 2002, (now U.S. Pat. No. 6,852,819), which is adivisional application of Ser. No. 09/207,203, filed Dec. 8, 1998, nowabandoned.

FIELD OF THE INVENTION

The present invention relates to an excellent stimuli-responsive polymerderivative which can be used for drug delivery system (DDS), chemovalve,various separating agents, catheter, artificial muscle, etc.

BACKGROUND OF THE INVENTION

In recent years, stimuli-responsive polymers have been widely used fordrug delivery system (DDS), various separating agents, catheter,artificial muscle, chemovalve, etc. and thus have been of growingimportance. For example, JP-A-8-103653 (The term “JP-A” as used hereinmeans an “unexamined published Japanese patent application”) discloses apolymer which changes in its higher order structure to swell or shrinkin an aqueous solution by the action of heat, light or by a change in pHor potential as a stimuli-responsive polymer. Specifically, acrylamideor methacrylamide derivatives such as poly-N-isopropylacrylamide,N,N-diethylacrylamide and N-isopropylmethacrylamide, and vinyletherssuch as vinyl methyl ether are disclosed as a polymer having an uppercritical solution temperature (UCST) or a lower critical solutiontemperature (LCST) with respect to water, which swells or shrinks inresponse to a temperature change.

Although these known polymers which swell or shrink in response to atemperature change are described as having an upper critical solutiontemperature (UCST) or a lower critical solution temperature (LCST), theyall have, in fact, a lower critical solution temperature (LCST). Inother words, at a temperature of not lower than the lower criticalsolution temperature, these polymers reversibly undergo agglomeration ofpolymers that renders themselves insoluble in water. On the contrary, ata temperature of not higher than the lower critical solutiontemperature, these polymers can be dissolved in water. For example,poly-N-isopropylacrylamide (PNIPAM), which is applied to DDS, etc. atpresent, has a lower critical solution temperature of 32° C. in anaqueous solution. When this polymer is allowed to gel, it reversiblyundergoes swelling and shrinkage depending on the temperature developedby heat.

A polymer having a lower critical solution temperature (LSCT) shrinks ata predetermined temperature or higher and thus is disadvantageous inthat it can be hardly adjusted so as to meet the demand for shrinkage atlow temperature (preferably not higher than the body temperature) in theapplication to DDS, separating agent, etc.

However, all these known thermo-responsive polymers such aspoly-N-isopropylacrylamide are stimuli-responsive polymers having alower critical solution temperature (LCST) which respond only to thermalstimulation. Thus, these thermo-responsive polymers can neither switch alower critical solution temperature to an upper critical solutiontemperature (UCST) nor have, in a single compound, both functions ofcausing their reversible dissolution and precipitation depending on thehydrogen ion concentration, when they respond to heat.

On the other hand, as the polymer which changes in its higher orderstructure by a pH change there is known a polyacrylic acid orpolymethacrylic acid. However, these compounds contain carboxylic acid,which has electric charge, and thus are disadvantageous in that aseparating agent comprising such a polymer adsorbs compounds other thandesired compounds (non-specific adsorption) and thus cannot provideefficient separation and purification.

If a composite stimuli-responsive polymer which can switch between alower critical solution temperature (LCST) and an upper criticalsolution temperature (UCST) or have, in a single compound, bothfunctions of causing its reversible dissolution and precipitationdepending on the hydrogen ion concentration can be obtained, the abovedescribed adjustment can be easily conducted. The appearance of such apolymer has been desired particularly in an art requiring fineadjustment because such a thermo-responsive polymer can be more widelyused.

Further, if used as a separating agent for protein inert to heat, etc.,the conventional polymer agglomerates when heated, causing denaturationof protein.

Moreover, if the polymer is used as DDS (e.g., chemical-releasingcapsule) by encapsulating a chemical in its gel, it is necessary thatthe affected part be cooled to allow the gel to swell and release thechemical upon releasing. However, it is practically easy to raise,rather than cool, the temperature of the affected part.

Further, if a thermo-responsive polymer is used as DDS, it needs toexhibit an upper critical solution temperature (UCST) in physiologicalsaline. In this respect, an interpenetration polymer network (IPNa) ofpolyacrylic acid and polyacryloyl glycinamide is known as athermo-responsive polymer which exhibits an upper critical solutiontemperature (UCST) in an aqueous solution (Makromol. Chem., RapidCommun. 13, 557-581 (1992)). However, this polymer does not exhibit anyupper critical solution temperature (UCST) in physiological saline.

Therefore, the appearance of a thermo-responsive polymer whichagglomerates when heated in an aqueous solution and exhibits an uppercritical solution temperature (UCST) even in physiological saline hasbeen desired.

SUMMARY OF THE INVENTION

An object of the present invention is to provide solution to the abovedescribed problems.

A first object of the present invention is to provide astimuli-responsive polymer which exhibits an upper critical solutiontemperature (UCST) or a stimuli-responsive polymer which undergoesreversible dissolution and precipitation depending on the hydrogen ionconcentration or an addition of a solvent.

A second object of the present invention is to provide athermo-responsive polymer which exhibits both a lower critical solutiontemperature (LCST) and an upper critical solution temperature (UCST).

A third object of the present invention is to provide a compositestimuli-responsive polymer which can switch between a lower criticalsolution temperature (LCST) and an upper critical solution temperature(UCST) or can have, in a single compound, both functions of causing itsreversible dissolution and precipitation depending on the hydrogen ionconcentration.

A fourth object of the present invention is to provide athermo-responsive polymer which agglomerates when heated in an aqueoussolution and exhibits an upper critical solution temperature (UCST) evenin physiological saline.

Further, the present invention also concerns a simple process forproducing an N-acyl(meth)acrylamide derivative which can be used as amonomer for the stimuli-responsive polymer, a process for the productionof an intermediate thereof, and an intermediate thus produced.

A first aspect of the present invention concerns the following polymerderivatives:

1-1) A stimuli-responsive polymer derivative having an upper criticalsolution temperature utilizing keto-enol tautomerization.

1-2) A stimuli-responsive polymer derivative utilizing keto-enoltautomerization which undergoes phase transition by a change in hydrogenion concentration or by an addition of an organic solvent.

1-3) The stimuli-responsive polymer derivative according to the above1-1) or 1-2), which comprises as a polymerizable component a monomerrepresented by the following general formula (1):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alky, alkoxyl, alkylamino, aryl or heterocyclic groupwhich may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or methyl group;and X and X′ each independently represents an oxygen atom, sulfur atom,selenium atom or tellurium atom.

1-4) The stimuli-responsive polymer derivative according to the above1-1) or 1-2), which comprises as copolymerizable components ahydrophilic or hydrophobic monomer and a monomer represented by generalformula (1).

1-5) The stimuli-responsive polymer derivative according to the above1-3), which comprises as a polymerizable component a monomer representedby the following general formula (7):

1-6) A stimuli-responsive separating agent comprising astimuli-responsive polymer derivative according to any one of the above1-1) to 1-5).

The invention paid attention to strong hydrogen bonding propertiesrepresented by peptide bond and reversible keto-enol tautomerization. Onthe supposition that a thermo-responsive polymer having an uppercritical solution temperature (UCST) can be obtained using keto-enolswitching as shown in the following reaction formula A, the presentinvention has been worked out.

In other words, the chemical reaction was designed using a computerizedmethod for the calculation of molecular orbital such that enolationoccurs at a high temperature to effect hydration and conversion to ketoform occurs at a low temperature to effect agglomeration by hydrogenbond. As a result, it was found that the above described design allowsthe appearance of an upper critical solution temperature (UCST). Moreparticularly, it is preferred to synthesize a compound in which the sitehaving a peptide bond is thermodynamically stable in its keto form.

Further, the above described theory gives a finding that the utilizationof keto-enol tautomerization makes it possible to make the abovedescribed keto-enol switching (reversible conversion between keto formand enol form) effectively not only by a thermal change but also by achange in hydrogen ion concentration or an addition of an organicsolvent, i.e., to obtain a stimuli-responsive polymer which reversiblyrepeats swelling and shrinkage in accordance with a change in hydrogenion concentration or an addition of an organic solvent without raisingor lowering the temperature.

The stimuli-responsive polymer derivative of the present aspect of thepresent invention can be effectively applied to the separation, fixing,calibration and control of various substances. In particular, thestimuli-responsive polymer derivative exhibits an upper criticalsolution temperature (UCST), i.e., agglomerates when the temperaturelowers or reversibly repeats swelling and shrinkage in accordance with achange in hydrogen ion concentration or an addition of an organicsolvent without raising or lowering the temperature. Accordingly, it isparticularly useful for the separation, purification, fixing,calibration and control of substances which are desirably not to be in ahigh temperature atmosphere (protein such as biological product, enzymeand antibody).

A second aspect of the present invention concerns a copolymer derivativecomprising a monomer component having a lower critical solutiontemperature (LCST) and a monomer component having an upper criticalsolution temperature (UCST).

As the monomer component having a lower critical solution temperature(LCST), a monomer represented by any one of the following generalformulae (2) to (5) can be used:

wherein R¹ represents a hydrogen atom or a methyl group; R² and R³ eachindependently represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R⁴ represents a C₁₋₁₀ straight-chain,branched or cyclic alkyl or alkylakoxyl group which may be halogenated;R⁶ represents a C₁₋₁₀ straight-chain, branched or cyclic alkyl, alkoxyl,alkylamino, aryl or heterocyclic group which may be halogenated; and nrepresents an integer of 4 or 5.

As the monomer component having an upper critical solution temperature(UCST), a monomer represented by the above described general formula (1)can be preferably used.

The stimuli-responsive polymer derivative of the present invention mayfurther comprise as a third component a hydrophilic or hydrophobiccopolymerizable monomer incorporated therein. The transition point ofthe stimuli-responsive polymer derivative can be controlled by theincorporation.

It can be presumed that the monomer component represented by the abovedescribed general formula (1) exhibits strong hydrogen bondingproperties represented by peptide bond and a reversible keto-enoltautomerization and has an upper critical solution temperature (UCST)developed by keto-enol switching as shown in the following reactionformula A:

In other words, the chemical reaction was designed using a computerizedmethod for the calculation of molecular orbital such that enolationoccurs at a high temperature to effect hydration and conversion to ketoform occurs at a low temperature to effect agglomeration by hydrogenbond. As a result, it was found that the above described design allowsthe appearance of an upper critical solution temperature (UCST). Moreparticularly, it is preferred to synthesize a compound in which the sitehaving a peptide bond is thermodynamically stable in its keto form.

The thermo-responsive copolymer derivative of the present aspect of thepresent invention can be effectively applied to the separation, fixing,calibration and control of various substances. In particular, thethermo-responsive copolymer derivative of the present aspect of thepresent invention has both an upper critical solution temperature (UCST)and a lower critical solution temperature (LCST). Accordingly, it can beeffectively used for the separation, purification, fixing, calibrationor control of substances the working temperature of which can be hardlypredetermined (protein such as biological product, enzyme and antibody).Alternatively, it can be effectively used for chemovalve.

A third aspect of the present invention concerns a compositestimuli-responsive polymer derivative having a lower critical solutiontemperature (LCST) comprising at least one monomer component representedby the following general formula (6):

wherein R⁵ represents a hydrogen atom or a methyl group; and R¹represents a hydrogen atom or a C₁₋₁₀ straight-chain, branched or cyclicalkyl, alkoxyl, alkylamino, aryl or heterocyclic group which may behalogenated. The compounds of general formula (6) corresponds tocompounds of general formula (1) wherein R² represents a single bond, R³and R⁴ each represents a hydrogen atom, and X and X′ each represents anoxygen atom.

The above described polymer derivative acts as a thermo-responsivepolymer which exhibits a lower critical solution temperature (LCST) inan aqueous solution. The lower critical solution temperature (LCST) canbe reversibly changed by the hydrogen ion concentration. In other words,the above described polymer derivative shows a composite stimulationresponse, that is, individually responds to heat and pH when stimulatedby pH.

Further, when put in an aqueous solution having a small amount of anorganic solvent added thereto, this thermo-responsive polymer loses thelower critical solution temperature (LCST), which has appeared so far,but exhibits an upper critical solution temperature (UCST). In otherwords, when stimulated by an organic solvent added, thisthermo-responsive polymer undergoes conversion of lower criticalsolution temperature to upper critical solution temperature.

It is particularly preferred that the stimuli-responsive polymerderivative of the present aspect of the present invention be a copolymerderivative comprising as a copolymerizable component at least onemonomer component which is hydrophilic or hydrophobic with respect tothe monomer component represented by general formula (6).

The term “monomer component which is hydrophilic or hydrophobic withrespect to the monomer component represented by general formula (6)” asused herein is intended to mean, if the monomer of general formula (6)is hydrophobic, a monomer component which is more hydrophilic than thehydrophobic monomer component of general formula (6), and if the monomerof general formula (6) is hydrophilic, a monomer component which is morehydrophobic than the hydrophilic monomer component represented bygeneral formula (6). The hydrophilic or hydrophobic monomer may be amonomer component represented by general formula (6) so far as it ishydrophilic or hydrophobic with respect to the one monomer componentrepresented by general formula (6). In this case, the hydrophilic orhydrophobic monomer contains two or more monomer components representedby general formula (6).

Accordingly, a preferred embodiment of the present aspect of the presentinvention is a copolymer further comprising at least one monomercomponent which is hydrophilic or hydrophobic with respect to onemonomer component represented by general formula (6) (including amonomer component represented by general formula (6)).

The content of the above described hydrophilic or hydrophobic monomer ispreferably from 1 to 70% by weight, more preferably from 3 to 50% byweight based on the total weight of the polymer. When the content of theabove described hydrophilic or hydrophobic monomer falls within theabove defined range, the above described properties of the presentaspect of the present invention can be exerted particularly effectively.

The thermo-responsive polymer derivative of the present aspect of thepresent invention can be effectively applied to the separation, fixing,calibration or control of various substances. In particular, thethermo-responsive polymer derivative of the present aspect of thepresent invention is a composite stimuli-responsive polymer which hasboth an upper critical solution temperature (UCST) and a lower criticalsolution temperature (LCST) within various temperature ranges andresponds also to hydrogen ion concentration. Accordingly, it can beeffectively used for the separation, purification, fixing, calibrationor control of substances the working temperature of which can be hardlypredetermined (protein such as biological product, enzyme and antibody).Alternatively, it can be effectively used for chemovalve.

A fourth aspect of the present invention concerns a thermo-responsivepolymer derivative having an upper critical solution temperature (UCST)in an aqueous solution, which comprises at least one monomer componentrepresented by the following general formula (6) and at least onemonomer component selected from acrylamide and methacrylamide:

wherein R⁵ represents a hydrogen atom or methyl group; and R¹ representsa hydrogen atom or a C₁₋₁₀ straight-chain, branched or cyclic alkyl,alkoxyl, alkylamino, aryl or heterocyclic group which may behalogenated.

The thermo-responsive polymer derivative of the present aspect of thepresent invention is a copolymer derivative comprising at least onemonomer component represented by general formula (6) and at least onemonomer component selected from acrylamide and methacrylamide ascopolymerizable components.

In the present aspect of the present invention, the charged proportionof the monomer represented by general formula (6) is preferably from 0.1to 100% by weight, more preferably from 1 to 30% by weight, particularlyfrom 5 to 15% by weight based on the weight of acrylamide and/ormethacrylamide.

The thermo-responsive polymer derivative of the present aspect of thepresent invention may further comprise at least one hydrophilic orhydrophobic monomer component which is hydrophilic or hydrophobic withrespect to monomer component represented by general formula (6)(excluding acrylamide and methacrylamide) incorporated therein as acopolymerizable component as necessary. The term “monomer componentwhich is hydrophilic or hydrophobic with respect to the monomercomponent represented by general formula (6)” as used herein is intendedto mean, if the monomer of general formula (6) is hydrophobic, a monomercomponent which is more hydrophilic than the hydrophobic monomercomponent of general formula (6), and if the monomer of general formula(6) is hydrophilic, a monomer component which is more hydrophobic thanthe hydrophilic monomer component represented by general formula (6).The hydrophilic or hydrophobic monomer may be a monomer componentrepresented by general formula (6) so far as it is hydrophilic orhydrophobic with respect to the one monomer component represented bygeneral formula (6). In this case, the hydrophilic or hydrophobicmonomer contains two or more monomer components represented by generalformula (6).

The charged proportion of the above described hydrophilic or hydrophobicmonomer is preferably from 1 to 70% by weight, more preferably from 3 to50% by weight based on the total weight of the monomer componentrepresented by general formula (6) and acrylamide and/or methacrylamide.

The thermo-responsive polymer derivative of the present inventionexhibits an upper critical solution temperature (UCST) in an aqueoussolution, particularly physiological saline, and thus can be effectivelyapplied to the separation, fixing, calibration or control of varioussubstances. Accordingly, it can be effectively used for the separation,purification, fixing, calibration or control of substances the workingtemperature of which can be hardly predetermined (protein such asbiological product, enzyme and antibody). Alternatively, it can beeffectively used for chemovalve, drug delivery system (DSS), etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further described hereinafter.

In accordance with the first aspect of the present invention, asmentioned above, by properly making a molecular design using keto-enoltautomerization, a stimuli-responsive polymer derivative having an uppercritical solution temperature (UCST) or a stimuli-responsive polymerderivative which undergoes phase transition in accordance with a changein hydrogen ion concentration or an addition of an organic solvent canbe easily obtained.

For example, a polymer derivative containing a substituent componentrepresented by the following general formula (8) may be preferably used:

wherein R¹, X and X′ are the same as those defined in general formula(1), respectively. The preferred ranges thereof are also the same asthose described below with reference to general formula (1).

In the polymer derivative containing a substituent component representedby general formula (8) (hereinafter described with reference to the casewhere X and X′ each represents an oxygen atom for the simplification ofdescription), the amide bonding site shows reversible switch betweenketo form and enol form as shown in the following reaction formula B inaccordance with an application of heat, a change in the hydrogen ionconcentration, or with an addition of an organic solvent.

Further, the present inventors found that a polymer derivativecontaining a monomer represented by the above described general formula(1) as a polymer component is particularly effective for efficientreversible keto-enol conversion.

In general, a compound having an amide bond itself agglomerates due tostrong hydrogen bonding in an aqueous solution. A polyamide which takesa keto form in an aqueous solution is insoluble in water. However, itcan be presumed that this keto form is converted to an enol form due toheat or a change in hydrogen ion concentration to lose itsself-agglomeration effect, giving a water-soluble compound.

The monomer represented by general formula (1) is described in moredetail below.

In general formula (1), R¹ preferably represents a C₁₋₈ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino or phenyl group, morepreferably methyl, ethyl, propyl, isopropyl, phenyl, methoxy, propoxyl,isopropoxyl, methylamino or ethylamino group, particularly methyl,ethoxy or methylamino group. These groups may be substituted by ahalogen atom such as fluorine, bromine, chlorine and iodine.Particularly preferred substituents are fluorine atom and chlorine atom.

R² preferably represents a single bond or C₁₋₂ straight-chain orbranched alkylene group or halogenated alkylene group, particularly asingle bond. Preferred examples of substituents on the alkylene groupinclude fluorine atom and chlorine atom.

X and X′ each is preferably an oxygen atom or sulfur atom.

Examples of the monomer represented by general formula (1) includeN-acetylacrylamide, N-fluoroacetylacrylamide, N-propionylacrylamide,N-butanoylacrylamide, N-pentanoylacrylamide, N-hexanoylacrylamide,N-isobutanoylacrylamide, N-benzoylacrylamide,N-(3-fluorobenzoyl)acrylamide, N-(2,3-difluorobenzoyl)acrylamide,N-pyridylcarbonylacrylamide, N-pyrimidylcarbonylacrylamide,N-acetylmethacrylamide, N-fluoroacetylmethacrylamide,N-propionylmethacrylamide, N-butanoylmethacrylamide,N-pentanoylmethacrylamide, N-hexanoylmethacrylamide,N-isobutanoylmethacrylamide, N-benzoylmethacrylamide,N-(3-fluorobenzoyl)methacrylamide,N-(2,3-difluorobenzoyl)methacrylamide, N-pyridylcarbonylmethacrylamide,N-pyrimidylcarbonylmethacrylamide, N-acroyl-N′-methylurea,N-acroyl-N′-ethylurea, N-acroyl-N′-fluoromethylurea,N-acroyl-N′-difluoromethylurea, N-acroyl-N′-trifluoromethylurea,N-methacroyl-N′-methylurea, N-methacroyl-N′-ethylurea,N-methacroyl-N′-fluoromethylurea, N-methacroyl-N′-difluoromethylurea,N-methacroyl-N′-trifluoromethylurea, methyl N-acroylcarbamate, ethylN-acroylcarbamate, n-propyl N-acroylcarbamate, isopropylN-acroylcarbamate, n-butyl N-acroylcarbamate, isobutylN-acroylcarbamate, fluoromethyl N-acroylcarbamate, difluoromethylN-acroylcarbamate, trifluoromethyl N-acroylcarbamate,2,2,2-trifluoroethyl N-acroylcarbamate, methyl N-methacroylcarbamate,ethyl N-methacroylcarbamate, n-propyl N-methacroylcarbamate, isopropylN-methacroylcarbamate, n-butyl N-methacroylcarbamate, isobutylN-methacroylcarbamate, t-butyl N-methacroylcarbamate, fluoromethylN-methacroylcarbamate, difluoromethyl N-methacroylcarbamate,trifluoromethyl N-methacroylcarbamate, and 2,2,2-trifluoroethylN-methacroylcarbamate.

Specifically, homopolymerization of a monomer represented by generalformula (1) or copolymerization of a monomer represented by generalformula (1) with a hydrophilic or hydrophobic monomer makes it possibleto obtain thermo-responsive polymers having a UCST within varioustemperature ranges, pH-responsive polymers which respond to varioushydrogen ion concentrations or solvent-responsive polymers which respondto an addition of an organic solvent.

Further, copolymerization of a monomer represented by general formula(1) with a monomer for a thermo-responsive polymer having an LCST makesit possible to obtain a heat- and pH-responsive polymer which exhibitsboth heat response and pH response.

The thermo-responsive polymer having a UCST preferably exhibits an uppercritical solution temperature of from 0 to 50° C., particularly from 0to 38° C., if it is used as a separating agent.

Further, the switching range of the thermo-responsive polymer (range ofphase transition temperature) is preferably as narrow as possible. Inaccordance with the present invention, a thermo-responsive polymerhaving a practical switching range of higher than 10° C. can beobtained.

The organic solvent to be used for stimulation is not specificallylimited so far as it has some solubility in water. Specific examples ofthe organic solvent include methanol, ethanol, propanol, isopropanol,acetone, THF, dioxane, acetic acid, propionic acid, ethylene glycol, andpropylene glycol. Preferred among these organic solvents are methanol,ethanol, propanol, isopropanol, acetone, and THF. These organic solventscan efficiently accelerate the agglomeration of the stimuli-responsivepolymer, though depending on the kind of the stimuli-responsive polymer.

It was also found that the application of stimulation by an organicsolvent makes it possible to develop a keto-enol switching type heatresponse.

Further, the novel stimuli-responsive polymer derivative of the presentinvention is effective for the separation, fixing, calibration orcontrol of substances which are desirably not to be in a hightemperature atmosphere. It can be effectively applied to drug deliverysystem (DDS), various separating agents, catheter, artificial muscle,etc.

In particular, the stimuli-responsive polymer derivative of the presentinvention can contain a region having affinity for the target substanceand a region showing the above described stimulation response to providean effective stimuli-responsive separating material.

The stimuli-responsive separating material of the present invention maybe in any embodiment normally used in the art. The target substance isnot specifically limited. In practice, however, protein (e.g., enzyme,antibody, molecular chaperon, biological product), glycoprotein, nucleicacid, cell, artificial cell, synthetic polymer, etc. may be used.

The separating material of the present invention is a materialcontaining a region having a stimulation response utilizing the abovedescribed keto-enol tautomerization and a region having affinity for thetarget substance. The region having a stimulation response preferablycontains a substituent component represented by general formula (8).More particularly, it preferably contains a monomer componentrepresented by general formula (1) as a copolymerizable component.

The region having affinity for the target substance contains a componentwhich can be bonded to or adsorbed by the target substance. Moreparticularly, the polymer derivative of the invention preferablycontains a monomer component containing the above described componentwhich can be bonded to or adsorbed by the target substance as acomponent copolymerizable with the above described monomer componentshowing a stimulation response. The bonding of the monomer component tothe target substance does not necessarily need to be a covalent bond butmay be a bond utilizing ion complex or charge-transfer complex or bondutilizing a biochemical affinity.

Furthermore particularly, a protein such as antibody and enzyme, if any,can be bonded to the stimuli-responsive material (region havingaffinity) by making the use of the reactivity of a functional group suchas amino group and carboxyl group which is often contained in such aprotein. For example, if an amino group in a protein is used, a carboxylgroup may be incorporated in the stimuli-responsive material to producean amide bond by the following reaction formula:

-   -   Condensation agent        R—NH₂+HOOC—R′→R—NH—CO—R′+H₂O        wherein R represents a protein; and R′ represents a        stimuli-responsive material

A method utilizing an aldehyde group and a method utilizing an epoxygroup as mentioned below may be used:

Further, if a carboxyl group in a protein is used, an amino group may beincorporated in the stimuli-responsive material to produce an amide bondby the following reaction formula:

-   -   Condensation agent        R—COOH+H₂N—R′→R—CO—NH—R′+H₂O

Further, an antibody may be incorporated in the stimuli-responsivematerial so that it is bonded to a protein as target substance. Thisoperation is preferably effected in phosphoric acid or trisbuffer havinga pH value in the vicinity of neutrality. The salt concentration may beproperly predetermined depending on the purpose.

Moreover, a particulate magnetic material may be bonded to thestimuli-responsive material to effect complexing. In this arrangement, astimuli-responsive material to which the target substance has beenbonded or by which the target has been adsorbed can be more efficientlyagglomerated by the use of a magnet or the like during separation.

The target substance which has been bonded to or adsorbed by thestimuli-responsive material of the present invention can be easilyeluted out by any of (1) raising the salt concentration, (2) changingthe pH value (rendering the solution acidic or alkaline), (3) adding aninhibitor, substrate, etc., (4) adding a modifier such as urea and SDS,(5) adding an organic solvent, metal ion, etc. and (6) changing thetemperature.

More particularly, the stimuli-responsive separating material of thepresent invention can be applied to medicine for detecting residualagricultural chemical or diagnostic medicine and can be effectively usedfor the activation or maintenance of biological reaction by separationof biological products such as microorganisms and product of cellculture or fixing of enzyme or molecular chaperon.

In accordance with the second aspect of the present invention, asmentioned above, a thermo-responsive polymer derivative having both alower critical solution temperature (LCST) and an upper criticalsolution temperature (UCST) can be obtained by copolymerizing at leastone monomer component having a lower critical solution temperature(LCST) with at least one monomer component having an upper criticalsolution temperature (UCST).

In the second aspect of the present invention, as the monomer componenthaving a lower critical solution temperature (LCST) there may bepreferably used a monomer component represented by any of the abovedescribed general formulae (2) to (5).

Particularly preferred embodiments of the various substituents ingeneral formulae (2) to (5) are described below.

R¹ preferably is a hydrogen atom or methyl group. R² and R³ each ispreferably a hydrogen atom, methyl group, ethyl group, propyl group,isopropyl group, n-butyl group, isobutyl group or tert-butyl group. R⁴is preferably a methyl group. R⁵ is preferably a methyl group, ethylgroup, propyl group, isopropyl group, n-butyl group, isobutyl group ortert-butyl group.

Specific examples of the monomer component represented by generalformula (2) include N-methylacrylamide, N-ethylacrylamide,N-cyclopropylacrylamide, N-isopropylacrylamide, N-n-propylacrylamide,N-tert-butylacrylamide, N-sec-butylacrylamide, N-n-butylacrylamide,N-methylmethacrylamide, N-ethylmethacrylamide,N-cyclopropylmethacrylamide, N-isopropylmethacrylamide,N-n-propylmethacrylamide, N-tert-butylmethacrylamide,N-sec-butylmethacrylamide, N-n-butylmethacrylamide,N,N-dimethylacrylamide, N,N-diethylacrylamide,N,N-dimethylmethacrylamide, N,N-diethylmethacrylamide,N-methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide,N-methyl-N-n-propylacrylamide, N-methyl-N-ethylmethacrylamide,N-methyl-N-isopropylmethacrylamide, N-methyl-N-n-propylmethacrylamide,N,N-diisopropylacrylamide, N,N-di-n-propylacrylamide,N,N-diisopropylmethacrylamide, and N,N-di-n-propylmethacrylamide.

Specific examples of the monomer component represented by generalformula (3) include methyl vinyl ether, and methoxy ethyl vinyl ether.

Specific examples of the monomer component represented by generalformula (4) include N-vinylacetamide, N-vinylpropionamide,N-vinylbutyrylamide, and N-vinyl isobutyrylamide.

Specific examples of the monomer component represented by generalformula (5) include N-acetylacrylamide, N-fluoroacetylacrylamide,N-propionylacrylamide, N-butanoylacrylamide, N-pentanoylacrylamide,N-hexanoylacrylamide, N-isobutanoylacrylamide, N-benzoylacrylamide,N-(3-fluorobenzoyl)acrylamide, N-(2,3-difluorobenzoyl)acrylamide,N-pyridylcarbonylacrylamide, N-pyrimidylcarbonylacrylamide,N-acetylmethacrylamide, N-fluoroacetylmethacrylamide,N-propionylmethacrylamide, N-butanoylmethacrylamide,N-pentanoylmethacrylamide, N-hexanoylmethacrylamide,N-isobutanoylmethacrylamide, N-benzoylmethacrylamide,N-(3-fluorobenzoyl)methacrylamide,N-(2,3-difluorobenzoyl)methacrylamide, N-pyridylcarbonylmethacrylamide,N-pyrimidylcarbonylmethacrylamide, N-acroyl-N′-methylurea,N-acroyl-N′-ethylurea, N-acroyl-N′-fluoromethylurea,N-acroyl-N′-difluoromethylurea, N-acroyl-N′-trifluoromethylurea,N-methacroyl-N′-methylurea, N-methacroyl-N′-ethylurea,N-methacroyl-N′-fluoromethylurea, N-methacroyl-N′-difluoromethylurea,N-methacroyl-N′-trifluoromethylurea, methyl N-acroylcarbamate, ethylN-acroylcarbamate, n-butyl N-acroylcarbamate, isopropylN-acroylcarbamate, n-butyl N-acroylcarbamate, isobutylN-acroylcarbamate, t-butyl N-acroylcarbamate, fluoromethylN-acroylcarbamate, difluoromethyl N-acroylcarbamate, trifluoromethylN-acroylcarbamate, 2,2,2-trifluoroethyl N-acroylcarbamate, methylN-methacroylcarbamate, ethyl N-methacroylcarbamate, n-butylN-methacroylcarbamate, isopropyl N-methacroylcarbamate, n-butylN-methacroylcarbamate, isobutyl N-methacroylcarbamate, t-butylN-methacroylcarbamate, fluoromethyl N-methacroylcarbamate,difluoromethyl N-methacroylcarbamate, trifluoromethylN-methacroylcarbamate, and 2,2,2-trifluoroethyl N-methacroylcarbamate.

On the other hand, as the monomer component having an upper criticalsolution temperature (UCST) there may be preferably used a monomercontaining a substituent component represented by general formula (8).

In the second aspect of the present invention, the composition ratio ofthe monomer component having a lower critical solution temperature(LCST) to the monomer component having an upper critical solutiontemperature (UCST) is not specifically limited but can be properlypredetermined depending on the purpose. In general, the weight ratio ofthe monomer component having a lower critical solution temperature tothe monomer component having an upper critical solution temperature ispreferably from 2:1 to 1:5.

The molecular weight of the polymer derivative of the second aspect ofthe present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. The molecularweight of the polymer derivative is normally from about 10² to 10⁶,preferably from about 10³ to 10⁵.

Further, in the second aspect of the present invention, thecopolymerization of a monomer component having a lower critical solutiontemperature (LCST) and a monomer component having an upper criticalsolution temperature (UCST) further with a hydrophilic or hydrophobicmonomer makes it possible to obtain thermo-responsive polymers whichexhibit a lower critical solution temperature (LCST) and an uppercritical solution temperature (UCST) within various temperature ranges.

The hydrophilic or hydrophobic monomer to be used herein is notspecifically limited. Various compounds may be used as such. Specificexamples of the hydrophilic monomer include acrylamide, allylamine,hydroxylethyl (meth)acrylate, and glycerin mono(meth)acrylate. Specificexamples of the hydrophobic monomer include ester (meth)acrylate, vinylchloride, vinylidene chloride, and styrene.

Further, the switching range (range of transition temperature) ispreferably as narrow as possible. In accordance with the second aspectof the present invention, a thermo-responsive polymer having a practicalswitching range of not higher than 10° C. can be obtained.

Further, the novel stimuli-responsive polymer derivative of the secondaspect of the present invention is effective for the separation, fixing,calibration or control of substances which are desirably not to be in ahigh temperature atmosphere. It can be effectively applied to drugdelivery system (DDS), various separating agents, catheter, artificialmuscle, chemovalve, etc.

A stimuli-responsive polymer derivative according to the third aspect ofthe present invention can be obtained, as mentioned above, bypolymerizing at least one monomer component represented by generalformula (6) or copolymerizing at least one monomer component representedby general formula (6) with at least one monomer component which ishydrophilic or hydrophobic with respect to the monomer component.

The monomer component represented by general formula (6) is described indetail below.

Preferred examples and particularly preferred examples of R′ in generalformula (6) include those described with reference to R¹ in generalformula (1). Specific examples of the monomer represented by generalformula (6) include those described with reference to general formula(1).

The hydrophilic or hydrophobic monomer to be additionally incorporatedas a copolymerizable component in the third aspect of the presentinvention cannot be unequivocally defined because it is hydrophilic orhydrophobic with respect to one monomer component represented by generalformula (6). Besides the monomer of general formula (1),(meth)acrylamide and (meth)acrylic acid may be used as hydrophilicmonomers and ester (meth)acrylate, vinyl chloride, vinylidene chloride,and styrene may be used as hydrophobic monomers.

In the third aspect of the present invention, as mentioned above, theincorporation of a monomer component represented by general formula (6)and optionally a monomer component which is hydrophilic or hydrophobicwith respect to the monomer component represented by general formula (6)makes it possible to obtain polymer derivatives having various lowercritical solution temperatures (LCST). The polymer derivative of thethird aspect of the present invention loses its transition point in anacidic or alkaline solution having a predetermined or higher acidity oralkalinity, e.g., aqueous solution of caustic soda having a normality ofnot less than 0.1 N, though depending on the kind of the monomercomponent used.

The organic solvent to be added to water to develop an upper criticalsolution temperature (UCST) in the third aspect of the present inventionis not specifically limited so far as it has solubility in water.Specific examples of the organic solvent employable herein includemethanol, ethanol, propanol, isopropanol, acetone, THF, dioxane, aceticacid, propionic acid, ethylene glycol, and propylene glycol.

Preferred among these organic solvents are methanol, ethanol, propanol,isopropanol, acetone, and THF. These organic solvents can efficientlyaccelerate the agglomeration of the stimuli-responsive polymer.

The amount of the organic solvent to be added depends on the kind of thestimuli-responsive polymer. In practice, however, it may be normallyfrom about 5 to 50% by weight so that the lower critical solutiontemperature (LCST) disappears while a lower critical solutiontemperature (LCST) appears.

The molecular weight of the polymer derivative of the third aspect ofthe present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. In practice,however, the weight-average molecular weight of the polymer derivativeis normally from about 10² to 10⁶, preferably from about 10³ to 10⁵.

In the third aspect of the present invention, the switching range of thethermo-responsive polymer (range of phase transition temperature) ispreferably as narrow as possible. In accordance with the third aspect ofthe present invention, a thermo-responsive polymer having a practicalswitching range of not higher than 10° C. can be obtained.

The novel stimuli-responsive polymer derivative of the second aspect ofthe present invention is effective for the separation, fixing,calibration or control of substances which are desirably not to be in ahigh temperature atmosphere. It can be effectively applied to drugdelivery system (DDS), various separating agents, catheter, artificialmuscle, chemovalve, etc.

A thermo-responsive polymer derivative according to the fourth aspect ofthe present invention can be obtained, as described above, bycopolymerizing at least one monomer component represented by generalformula (6) with at least one monomer component selected from acrylamideand methacrylamide and optionally the above described hydrophilic orhydrophobic monomer component.

Preferred examples and particularly preferred examples of R¹ in generalformula (6) include those described with reference to R¹ in generalformula (1).

Specific examples of the monomer represented by general formula (6)include those described with reference to general formula (1), andN-formylacrylamide and N-formylmethacrylamide.

The hydrophilic or hydrophobic monomer to be additionally incorporatedas a copolymerizable component in the fourth aspect of the presentinvention cannot be unequivocally defined because it is hydrophilic orhydrophobic with respect to one monomer component represented by generalformula (6). Besides the monomer of general formula (6), acrylamide andmethacrylamide, (meth)acrylic acid, etc. may be used as hydrophilicmonomers and ester (meth)acrylate, vinyl chloride, vinylidene chloride,and styrene may be used as hydrophobic monomers.

The molecular weight of the polymer derivative of the fourth aspect ofthe present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. In practice,however, the weight-average molecular weight of the polymer derivativeis normally from about 10² to 10⁶, preferably from about 10³ to 10⁵.

The thermo-responsive polymer having a UCST preferably exhibits an uppercritical solution temperature of from 0 to 50° C., particularly from 0to 38° C., if it is used as a separating agent.

In the fourth aspect of the present invention, the switching range ofthe thermo-responsive polymer (range of phase transition temperature) ispreferably as narrow as possible. In accordance with the fourth aspectof the present invention, a thermo-responsive polymer having a practicalswitching range of not higher than 10° C. can be obtained.

The novel stimuli-responsive polymer derivative of the fourth aspect ofthe present invention is effective for the separation, fixing,calibration or control of substances which are desirably not to be in ahigh temperature atmosphere. It can be effectively applied to drugdelivery system (DDS), various separating agents, catheter, artificialmuscle, chemovalve, etc.

In particular, the stimuli-responsive polymer derivative of the presentinvention can contain a region having affinity for the target substanceand a region showing the above described stimulation response to providean effective stimuli-responsive separating material orchemical-releasing capsule. Chemical-releasing capsules are formulationswhich release a chemical enclosed therein controllably with reversibleswelling and shrinkage due to a temperature or pH change. Theseformulations have been spotlighted as intelligent formulations which cangive a chemical in a required amount as necessary.

The stimuli-responsive separating material and chemical-releasingcapsule of the present invention may be in any embodiment normally usedin the art. The target substance is not specifically limited. Inpractice, however, protein (e.g., enzyme, antibody, molecular chaperon,biological product), glycoprotein, nucleic acid, cell, artificial cell,synthetic polymer, various chemicals (e.g., carcinostatic such asadriamycin, taxol), etc. may be used.

The separating material and chemical-releasing capsule of the presentinvention are materials having a region showing the above describedstimulation response and a region having affinity for the targetsubstance. The region showing a stimulation response may contain asubstituent component represented by general formula (6).

Further, the thermo-responsive polymer of the fourth aspect of thepresent invention, if incorporated in a chemical-releasing capsule, ispreferably provided in the form of a thermo-responsive hydrogelcontaining at least one monomer component represented by general formula(6), at least one monomer component selected from acrylamide andmethacrylamide, and a crosslinking agent as a copolymerizable componentwhich is used as a chemical-releasing capsule.

As the above described crosslinking agent there is preferably used acompound terminated by double bond at both ends thereof. Examples ofsuch a compound include N,N′-methylenebisacrylamide, divinylbenzene,divinylsulfone, diallyl carbinol, divinylether, and 1,5-hexadiene.

A process for simply producing an N-acyl(meth)acrylamide derivativewhich can be used as a monomer of a stimuli-responsive polymer, aprocess for producing an intermediate thereof and an intermediate thusproduced are described below.

To date, several methods for the synthesis of N-acyl(meth)acrylamidehave been developed. However, these synthesis methods leave something tobe desired in yield and productivity. These synthesis methods and theirproblems will be described hereinafter.

The reaction of acrylamide and ketene gas as starting materialsrepresented by the following reaction formula (J. A. C. S., vol. 23, pp.915-916 (1958)) gives a good yield but requires the use of ketene gas,which is very toxic.

The reaction of acrylamide and an acid anhydride as starting materialsrepresented by the following reaction formula (JP-B-37-9212 (The term“JP-B” as used herein means an “examined Japanese patent publication”))produces Michael adducts besides N-acetylated compounds and thus gives apoor yield.

The reaction of acrylamide and an acid chloride represented by thefollowing reaction formula (U.S. Pat. No. 852,460) gives muchby-products and hence a poor yield.

In order to solve the above described problems, a simple productionprocess which gives a good yield has been desired.

As a result of the extensive studies made by the present inventors,simple production processes were found as described below.

That is, an N-acyl(meth)acrylamide derivative can be simply produced byreacting:

an isocyanate represented by general formula (9):

wherein R₁ represents a hydrogen atom or a methyl group; and R₂ and R₃each independently represents a hydrogen atom or a C₁₋₁₀ straight-chainor branched alkyl group which may be halogenated; with

an organic metal compound represented by the following general formula(10):

wherein M represents an alkaline metal or a halogenated alkaline earthmetal; the ring A represents a cyclohexane ring, cyclopentane ring,cyclopentadiene ring, pyridine ring, pyrimidine ring or benzene ring; nrepresents a positive number of from 0 to 4; and R₄, R₅ and R₆ eachindependently represents a hydrogen atom, a C₁₋₁₀ optionally halogenatedstraight-chain or branched alkyl group or halogen atom directlyconnected to the ring A or M, with the proviso that if n is 0, R₄ isneither a hydrogen atom nor a halogen atom, to thereby produce anN-acyl(meth)acrylamide derivative represented by general formula (11):

wherein R₁ to R₆, the ring A and n are as defined above can be simplyproduced.

In other words, by using an isocyanate represented by general formula(9) instead of the conventional acrylamide as a starting material andreating it with an organic metal reagent represented by general formula(10), the desired N-acyl(meth)acrylamide derivative can be simplyobtained in a high yield.

In the isocyanate represented by general formula (9), R₁ to R₃preferably each represents a hydrogen atom or methyl group.

Isocyanates represented by general formula (9) and organic metalcompounds represented by general formula (10) are all commerciallyavailable or may be easily produced from commercially availablecompounds by known methods.

The solvent to be used in the above described production process is notspecifically limited so far as it has no adverse effects on thereaction. Various solvents may be used so far as they do not react witha nucleophilic reagent. Such a solvent may be selected from aliphatichydrocarbon solvents such as cyclohexane, hexane and heptane, aromatichydrocarbon solvents such as benzene and toluene, halogenatedhydrocarbon solvents such as 1,2-dichloroethane, chloroform and carbontetrachloride and ether solvents such as diethyl ether, dioxane andtetrahydrofurane (THF). These solvents may be used singly or inadmixture.

The reaction is effected normally at a temperature of from −78° C. to70° C., preferably from −40° C. to 35° C. The reaction time is notspecifically limited. In practice, however, the reaction may beterminated when it ends in accordance with ordinary method. In general,the reaction time ranges from several minutes to 24 hours.

The compound represented by general formula (11) obtained according tothe present invention may be effectively used as a monomer component ofstimuli-responsive polymer which swells or shrinks due to a temperatureor pH change or an addition of a solvent or polymer such as plasticmodifier optionally together with other copolymerizable components.Further, analogues of this compound may be used as herbicides (see U.S.Pat. No. 852,460).

SYNTHESIS EXAMPLE 1-1 Synthesis of N-acetyl methacrylamide

10 ml of methacroyl isocyanate was dissolved in 50 ml of THF in a flask.To the solution was then added dropwise 35 ml of a 3 mol/l THF solutionof methyl magnesium bromide at a temperature of −20° C. in an atmosphereof nitrogen. After the termination of the dropwise addition, the mixturewas then stirred at room temperature for 1 hour. To the mixture werethen added 100 ml of a 2 N hydrochloric acid and 100 ml of ethyl acetatesequentially. The resulting organic phase was then washed twice withsaturated brine. The solvent was then distilled off under reducedpressure. The residue thus obtained was then recrystallized from ethylacetate to obtain 5.3 g of a colorless crystal (yield: 48%). NMRanalysis gave a strong indication that the product is the desiredcompound.

COMPARATIVE SYNTHESIS EXAMPLE 1-1 Synthesis of N-acetyl methacrylamideby conventional method

23.7 g of acrylamide and 60 ml of triethylamine were dissolved in 100 mlof dichloromethane in a flask. To the solution was then added dropwise27.6 g of acetyl chloride at a temperature of −30° C. After thetermination of the dropwise addition, the mixture was then stirred at atemperature of 0° C. for 10 hours. Triethylamine hydrochloride thusprecipitated was then filtered off. The filtrate was then subjected todistillation under reduced pressure to remove the solvent therefrom. Theresidue thus obtained was then subjected to column chromatography withethyl acetate as a developing solvent and silica gel as a filler toobtain 1.5 g of the desired compound (yield: 4%).

SYNTHESIS EXAMPLE 1-2 Synthesis of N-benzoyl methacrylamide

2 ml of methacroyl isocyanate was dissolved in 20 ml of THF in a flask.To the solution was then added dropwise a 3 mol/l THF solution of phenyllithium at a temperature of −20° C. in an atmosphere of nitrogen. Afterthe termination of the dropwise addition, the mixture was then stirredat room temperature for 1 hour. To the mixture were then added 100 ml ofa 2 N hydrochloric acid and 100 ml of ethyl acetate sequentially. Theresulting organic phase was then washed twice with saturated brine. Thesolvent was then distilled off under reduced pressure. The residue thusobtained was then recrystallized from ethyl acetate to obtain 1.3 g of acolorless crystal (yield: 40%).

NMR analysis gave a strong indication that the product is the desiredcompound.

SYNTHESIS EXAMPLE 1-3 Synthesis of Other N-acyl(meth)acrylamideDerivatives

The isocyanates represented by general formula (9) and the organic metalcompound represented by general formula (10) set forth in the tablebelow were reacted in the same manner as in Synthesis Example 1-1. As aresult, the desired compound (11) was obtained in a yield set forth inthe table below.

TABLE 1 Compound of general formula (10) Ethyl Propyl Compound of Phenylmagnesium magnesium general formula (9) lithium bromide bromide Acroylisocyanate 55% 45% 43% Methacroyl 51% 47% 44% isocyanate

In accordance with the above described production process of the presentinvention, an N-acyl(meth)acrylamide derivative which can be used as amonomer for a stimuli-responsive polymer or modifier or as a startingmaterial of herbicide can be simply synthesized in a good yield.

Another production process is described below.

That is, an N-acyl(meth)acrylamide derivative can be simply produced byreacting:

an amide represented by the following general formula (12):

wherein R₁ represents a hydrogen atom or a methyl group; and R₂ and R₃each independently represents a hydrogen atom or a C₁₋₁₀ straight-chainor branched alkyl group which may be halogenated; with

a compound represented by the following general formula (13):

wherein R₄ represents a C₁₋₁₀ straight-chain or branched alkyl group ora C₅₋₆ cyclic alkyl, aryl or heterocyclic group, each of which may behalogenated, to thereby produce an enamine compound represented by thefollowing general formula (14):

wherein R₁ to R₄ are as defined above; and then

allowing the enamine compound to undergo hydrolysis under acidicconditions, to thereby produce an N-acyl(meth)acrylamide derivativerepresented by the following general formula (15):

wherein R₁ to R₄ are as defined above.

This process of the present invention is characterized by using anacrylamide as a starting material and reacting it with a reagentrepresented by general formula (13) to produce a novel enaminerepresented by general formula (14). Further, in accordance with thisprocess of the present invention, the enamine can be hydrolyzed underacidic conditions to simply produce the desired N-acyl(meth)acrylamidein a good yield.

In the acrylamide represented by general formula (12), R₁ to R₃ eachpreferably represent a hydrogen atom or a methyl group.

In the reagent represented by general formula (13), R₄ preferablyrepresents a methyl group, ethyl group, trifluoromethyl group,cyclohexyl group or phenyl group.

Acrylamides represented by general formula (12) and reagents representedby general formula (13) are all commercially available or may be easilyproduced from commercially available compounds by known methods.

In the synthesis of an enamine at the first stage, the reaction canproceed without any solvent. However, some solvent is preferably usedfrom the standpoint of operation efficiency and yield. The solventemployable herein is not specifically limited so far as it has noadverse effects on the reaction. Various solvents may be used. Such asolvent may be selected from aliphatic hydrocarbon solvents such ascyclohexane, hexane and heptane, aromatic hydrocarbon solvents such asbenzene and toluene, halogenated hydrocarbon solvents such as1,2-dichloroethane, chloroform and carbon tetrachloride and ethersolvents such as diethyl ether, dioxane and tetrahydrofurane (THF).These solvents may be used singly or in admixture.

The first stage of the reaction is effected normally at a temperature offrom 0° C. to 200° C., preferably from 40° C. to 80° C. The reactiontime for the first stage is not specifically limited. In practice,however, the reaction may be terminated when it ends in accordance withordinary method. In general, the reaction time ranges from 30 minutes to24 hours.

The hydrolysis reaction at the second stage can proceed without anysolvent. However, some solvent is preferably used from the standpoint ofoperation efficiency and yield. The solvent employable herein is notspecifically limited so far as it has no adverse effects on thereaction. A water-soluble solvent is preferably used. Examples of such asolvent include ether solvents such as dioxane and tetrahydrofurane(THF), alcohols such as methanol, ethanol, propanol and isopropanol andorganic acids such as acetic acid and propionic acid. These solvents maybe used singly or in admixture.

As the acidic substance to be used in the hydrolysis reaction there maybe used any acidic substance such as protonic acid, Lewis acid andorganic acid without any restriction so far as it has no adverse effectson the reaction. Examples of such an acidic substance includehydrochloric acid, sulfuric acid, nitric acid, iron chloride, copperchloride, zinc chloride, acetic acid, propionic acid and trifluoroaceticacid. These acidic substances may be used singly or in admixture.

The reaction at the second stage is effected normally at a temperatureof from 0° C. to 100° C., preferably from 10° C. to 30° C. The reactiontime for the second stage is not specifically limited. In practice,however, the reaction may be terminated when it ends in accordance withordinary method. In general, the reaction time ranges from 30 minutes to24 hours.

The compound represented by general formula (15) obtained according tothe present invention may be effectively used as a monomer component ofstimuli-responsive polymer which swells or shrinks due to a temperatureor pH change or an addition of a solvent, or polymer such as plasticmodifier optionally together with other copolymerizable components.Further, analogues of this compound may be used as herbicides (see U.S.Pat. No. 852,460).

SYNTHESIS EXAMPLE 2-1 Synthesis of N-acetyl acrylamide

31 g of acrylamide and 80 g of N,N-dimethylacetamide dimethylacetal weredissolved in 200 ml of THF in a flask. The mixture was then stirred at atemperature of 65° C. for 3 hours. The disappearance of the startingmaterials was then confirmed by gas chromatography. Thereafter, thesolvent was distilled off by an evaporator. The initial distillate wasdistilled off under reduced pressure to obtain 40 g of(N,N-dimethylacetamide)imine in the form of slightly yellowed liquid.

NMR analysis gave a strong indication that the product is the abovedescribed imine substance, as follows.

¹H-NMR analysis: δ2.25 (multi. 6H), δ3.10 (s. 3H), δ5.64 (multi. 1H),δ6.27 (multi. 2H).

The imine thus obtained was dissolved in a mixture of 200 ml of a 2 Nhydrochloric acid and 40 ml of acetic acid, and then stirred at roomtemperature for 4 hours. The disappearance of the imine as a startingmaterial was then confirmed by gas chromatography. Thereafter, to thesolution were added 100 ml of water and 100 ml of ethyl acetate. Theresulting organic phase was then washed with an aqueous solution ofsodium bicarbonate until it became neutral. The organic phase was thendried over magnesium sulfate. The aqueous phase was collected together,and then extracted with ethyl acetate. The resulting organic phase waswashed with an aqueous solution of sodium bicarbonate until it becameneutral, and then added to the first batch of organic phase which wasthen again dried.

The solvent was then distilled off by an evaporator. The residue wasthen subjected to column chromatography with silica gel produced byMerck and ethyl acetate as a developing solvent to remove unreactedacrylamide therefrom. The fraction thus obtained was concentrated, andthen recrystallized twice from ethyl acetate to obtain 20 g of thedesired compound having a purity of 99.6% in the form of white crystal(yield: 41%).

NMR analysis gave a strong indication that the product is N-acetylacrylamide as follows:

¹H-NMR analysis: δ2.47 (s. 3H), δ5.89 (tri. 1H), δ6.48 (d. 2H), δ7.27(s. 1H).

COMPARATIVE SYNTHESIS EXAMPLE 2-1 Synthesis of N-acetyl acrylamide byconventional method

23.7 g of acrylamide and 60 ml of triethylamine were dissolved in 100 mlof dichloromethane in a flask. To the solution was then added dropwise27.6 g of acetyl chloride at a temperature of −30° C. After thetermination of the dropwise addition, the mixture was then stirred at atemperature of 0° C. for 10 hours. Triethylamine hydrochloride thusprecipitated was then filtered off. The filtrate was then subjected todistillation under reduced pressure to remove the solvent therefrom. Theresidue thus obtained was then subjected to column chromatography withethyl acetate as a developing solvent and silica gel as a filler toobtain 1.5 g of the desired compound (yield: 4%).

SYNTHESIS EXAMPLE 2-2 Synthesis of N-acetyl methacrylamide

33 g of methacrylamide and 80 g of N,N-dimethylacetamide dimethylacetalwere dissolved in 200 ml of THF in a flask. The mixture was then stirredat a temperature of 65° C. for 3 hours. The disappearance of thestarting materials was then confirmed by gas chromatography. Thereafter,the solvent was distilled off by an evaporator. The initial distillatewas distilled off under reduced pressure to obtain 50 g of(N,N-dimethylacetamide)imine substance in the form of slightly yellowedliquid. NMR analysis gave a strong indication that the product is thedesired imine.

The imine thus obtained was dissolved in a mixture of 200 ml of a 2 Nhydrochloric acid and 40 ml of acetic acid, and then stirred at roomtemperature for 4 hours. The disappearance of the imine as a startingmaterial was then confirmed by gas chromatography. Thereafter, to thesolution were added 100 ml of water and 100 ml of ethyl acetate. Theresulting organic phase was then washed with an aqueous solution ofsodium bicarbonate until it became neutral. The organic phase was thendried over magnesium sulfate. The aqueous phase was collected together,and then extracted with ethyl acetate. The resulting organic phase waswashed with an aqueous solution of sodium bicarbonate until it becameneutral, and then added to the first batch of organic phase which wasthen again dried.

The solvent was then distilled off by an evaporator. The residue wasthen subjected to column chromatography with silica gel produced byMerck and ethyl acetate as a developing solvent to remove unreactedmethacrylamide therefrom. The fraction thus obtained was concentrated,and then recrystallized twice from ethyl acetate to obtain 30 g of thedesired compound having a purity of 99.8% in the form of white crystal(yield: 62%).

NMR analysis gave a strong indication that the product is N-acetylmethacrylamide as follows:

¹H-NMR analysis: δ2.00 (multi. 3H), δ2.50 (s. 3H), δ5.66 (qur. 1H),δ5.96 (d. 1H), δ9.41 (br. s. 1H).

In accordance with the above described production process of the presentinvention, an N-acyl(meth)acrylamide derivative which can be used as amonomer for a stimuli-responsive polymer or modifier or as a startingmaterial of herbicide can be simply synthesized via a novel enamine in ahigh yield.

The present invention described in greater detail with reference to thefollowing Examples and comparative Examples, but the present inventionshould not be construed as being limited thereto.

EXAMPLE 1-1 Synthesis of N-acetyl (Meth)acrylamide (Scheme a)

In an atmosphere of nitrogen gas, 30.5 g of acrylamide, 80 g ofN,N-dimethylacetamide dimethylacetal and 400 ml of THF were charged in aflask, and then stirred at a temperature of 65° C. for 3 hours. Thereaction solution thus obtained was concentrated under reduced pressure.The residue was subjected to simple distillation under a pressure of 1mmHg to obtain 45 g of an acroylimide. The acroylimide thus obtained wasdissolved in 100 ml of a 2 N hydrochloric acid, and then charged into aflask. To the solution was then added 20 ml of acetic acid. The mixturewas then stirred at room temperature for 4 hours. The reaction solutionwas then extracted with ethyl acetate. The resulting organic phase wasthen concentrated under reduced pressure. The residue was then subjectedto column chromatography with ethyl acetate as a solvent. The resultingfraction was then concentrated under reduced pressure. The residue wasthen recrystallized from ethyl acetate as a solvent to obtain 30 g of awhite crystal.

The above described synthesis procedure was followed except that 30.5 gof methacrylamide was used as a starting material. As a result, 32 g ofthe desired compound was obtained.

NMR analysis gave a strong indication that the product is the desiredcompound.

EXAMPLE 1-2 Synthesis and physical properties of poly-N-acetylacrylamide

In an atmosphere of nitrogen gas, 1.0 g of N-acetyl acrylamide and 10 mgof AIBN were dissolved in ethanol, and then charged into a flask whereit was then stirred at a temperature of 75° C. for 3 hours. The polymerthus precipitated was withdrawn by filtration, thoroughly washed withethanol, and then dried at room temperature under reduced pressure toobtain 850 mg of a white solid.

50 g of the polymer thus obtained was heated and dissolved in 5 ml of a10% ethanol solution, 20% ethanol solution and 30% ethanol solution,respectively, and then allowed to cool. In this manner, these polymersolutions were measured for transparent point upon heating in the formof uniform cloudy liquid. As a result, these polymer solutions showed atransparent point of 38.5° C., 39.4° C. and 41.9° C., respectively.After reaching the transparent point, these polymer solutions weremeasured for cohesion temperature upon cooling. As a result, thesepolymer solutions were observed to show USCT at 44.7° C., 45.2° C. and50.2° C., respectively. These polymer solutions reversibly underwentdissolution and precipitation many times at these temperatures.

The measurement of transition temperature was effected as calculated interms of visible light transmittance.

The transition temperature range (temperature range required until thetransmittance reached from 2% to 100% upon heating or from 98% to 0%upon cooling) was as very narrow as from 2 to 6° C., though depending onthe ethanol concentration.

EXAMPLE 1-3 Synthesis and physical properties of poly-N-acetylmethacrylamide

In an atmosphere of nitrogen gas, 1.0 g of N-acetyl methacrylamide and10 mg of AIBN were dissolved in ethanol, and then charged into a flaskwhere it was then stirred at a temperature of 75° C. for 3 hours. Thepolymer thus precipitated was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 810 mg of a white solid.

50 mg of the polymer thus obtained was then dissolved in 5 ml of a 1 Naqueous solution of sodium hydroxide. To the solution thus obtained wasthen added dropwise a 0.1 N hydrochloric acid. As a result, it wasconfirmed that the polymer thus obtained is a pH-responsive polymerwhich repeatedly undergoes dissolution at a pH value of not less than10.3 and precipitation at a pH value of not more than 10.3.

EXAMPLE 1-4 Synthesis and physical properties of N-acetyl methacrylamideand N-isopropyl acrylamide copolymer

In an atmosphere of nitrogen gas, 1.0 g of N-acetyl methacrylamide, 1.0g of N-isopropyl acrylamide and 10 mg of AIBN were dissolved in ethanol,and then charged into a flask where it was then stirred at a temperatureof 75° C. for 3 hours. The polymer thus precipitated was withdrawn byfiltration, thoroughly washed with ethanol, and then dried at roomtemperature under reduced pressure to obtain 1.1 g of a white solid.

50 mg of the polymer thus obtained was dissolved in 5 ml of a bufferhaving pH 1, buffer having pH 5, buffer having pH 7, buffer having pH 10and buffer having pH 12, respectively. In this manner, these polymersolutions were measured for its LCST. As a result, these polymersolutions showed LCSTs of 52° C., 48° C., 48° C., 35° C. and 32° C.,respectively. The transition temperature range (temperature rangerequired until the transmittance reached from 98% to 0%) was as verysharp as from 1.5 to 6° C., though depending on pH.

COMPARATIVE EXAMPLE 1-1 Synthesis and physical properties ofpoly-N-isopropyl acrylamide (PNIPAM)

In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamide and 5mg of AIBN were dissolved in ethylene glycol dimethyl ether, and thencharged into a flask where it was then stirred at a temperature of 75°C. for 3 hours. The reaction solution thus obtained was thenreprecipitated from a 10/1 mixture of cyclohexane and ethyl acetate toobtain 0.6 g of a white solid.

50 mg of the polymer thus obtained was dissolved in 5 ml of a bufferhaving pH 1, buffer having pH 5, buffer having pH 7, buffer having pH 10and buffer having pH 12, respectively. In this manner, these polymersolutions were measured for its LCST. As a result, these polymersolutions were confirmed to show little or no dependence of the LCST onpH and exhibit an LCST of about 30° C.

EXAMPLE 1-5 Synthesis of trifluoroethyl N-methacroylcarbamate (Scheme b)

4 ml of methacroyl isocyanate was dissolved in 50 ml of THF in a flask.To the solution was then added dropwise 10 ml of 2,2,2-trifluoroethanolat a temperature of −40° C. in an atmosphere of nitrogen. After thetermination of the dropwise addition, the mixture was then stirred atroom temperature for 1 hour. The solvent was then distilled off underreduced pressure. The residue was then subjected to silica gel columnchromatography with ethyl acetate as a developing solvent. The fractionthus obtained was concentrated, and then recrystallized from ethylacetate as a solvent to obtain 4.0 g of a white crystal.

NMR analysis gave a strong indication that the product is the desiredcompound as follows.

NMR analysis: δ2.00 (s, 3H), δ4.52 (qr, 3H), δ5.65 (s, 1H), δ5.96 (s,1H), δ8.68 (s, 1H).

EXAMPLE 1-6 Synthesis and physical properties of trifluoroethylpoly-N-methacroylcarbamate

In an atmosphere of nitrogen gas, 1.0 g of trifluoroethylN-methacroycarbamate and 10 mg of AIBN were dissolved in ethanol, andthen charged into a flask where it was then stirred at a temperature of75° C. for 3 hours. The polymer thus precipitated was withdrawn byfiltration, thoroughly washed with ethanol, and then dried at roomtemperature under reduced pressure to obtain 520 mg of a white solid.

50 mg of the polymer thus obtained was then dissolved in 5 ml of a 1 Naqueous solution of sodium hydroxide. To the solution thus obtained wasthen added dropwise a 0.1 N hydrochloric acid. As a result, it wasconfirmed that the polymer thus obtained is a pH-responsive polymerwhich repeatedly undergoes dissolution at a pH value of not less than10.5 and precipitation at a pH value of not more than 10.5.

EXAMPLE 1-7 Synthesis of N-methacroyl-N-methylurea (Scheme c)

15 ml of methacroyl isocyanate was dissolved in 100 ml of THF in aflask. To the solution was then added dropwise 100 ml of a 2 mol/l THFsolution of methylamine at a temperature of −40° C. in an atmosphere ofnitrogen. After the termination of the dropwise addition, the mixturewas then stirred at room temperature for 1 hour. The solvent was thendistilled off under reduced pressure. The residue was then subjected tosilica gel column chromatography with ethyl acetate as a developingsolvent. The fraction thus obtained was concentrated, and thenrecrystallized from a 10/1 mixture of ethyl acetate and ethanol as asolvent to obtain 12 g of a white crystal. NMR analysis gave a strongindication that the product is the desired compound.

EXAMPLE 1-8 Synthesis and physical properties ofpoly-N-methacroyl-N-methylurea

In an atmosphere of nitrogen gas, 1.0 g of N-methacroyl-N-methylurea and10 mg of AIBN were dissolved in ethanol, and then charged into a flaskwhere it was then stirred at a temperature of 75° C. for 3 hours. Thepolymer thus precipitated was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 880 mg of a white solid.

50 mg of the polymer thus obtained was then dissolved in 5 ml of a 1 Naqueous solution of sodium hydroxide. To the solution thus obtained wasthen added dropwise a 0.1 N hydrochloric acid. As a result, it wasconfirmed that the polymer thus obtained is a pH-responsive polymerwhich repeatedly undergoes dissolution at a pH value of not less than12.1 and precipitation at a pH value of not more than 12.1.

EXAMPLE 1-9 Preparation of Immunoglobulin G-Separating AdsorptiveMaterial

The separation of immunoglobulin G as target substance was examinedusing a stimuli-responsive polymer and a protein. As thestimuli-responsive polymer there was used a poly-N-acetyl acrylamide. Asthe protein there was used Protein A having a specific affinity.

1 g of N-acryloxy succinimide and 20 g of N-acetyl acrylamide were thensubjected to polymerization with AIBN as an initiator and ethyleneglycol dimethyl ether as a solvent at a temperature of 70° C. for 3hours. The solid thus precipitated was withdrawn by filtration,thoroughly washed with acetone, and then dried under reduced pressure toobtain 18 g of a copolymer.

The copolymer thus obtained and 5 g of Protein A were dissolved in 500ml of distilled water at a temperature of 37° C., and then stirred for12 hours. After the termination of the reaction, to the solution wasadded 10 ml of ethanol. The temperature of the aqueous solution wasadjusted to 15° C. As a result, a copolymer containing Protein A wasprecipitated. The copolymer thus obtained was withdrawn by filtration,and then thoroughly rinsed with 5° C. distilled water to obtain astimuli-responsive separating material containing Protein A.

In an atmosphere of nitrogen, 5 g of the stimuli-responsive separatingmaterial thus obtained was dissolved in 1,000 ml of a 5% aqueoussolution of mouse blood plasma at a temperature of 37° C., and thenstirred for 20 minutes. To the solution was then added 20 ml of ethanol.The solution was then cooled to a temperature of 10° C. to causeprecipitation. The precipitate thus obtained was then rinsed withsaturated brine. The wash water was then analyzed by high-performanceliquid chromatography. As a result, it was confirmed that immunoglobulinG having a purity of 92% had been obtained.

In accordance with the first aspect of the present invention, astimuli-responsive polymer which exhibits an upper critical solutiontemperature (UCST) or a stimuli-responsive polymer which undergoesreversible dissolution and precipitation depending on the hydrogen ionconcentration or an addition of solvent can be obtained.

Further, the use of the above described stimuli-responsive polymer makesit possible to obtain an excellent stimuli-responsive separatingmaterial particularly effective for the separation of a target substancewhich is desirably not to be in a high temperature atmosphere.

EXAMPLE 2-1 Synthesis and physical properties of copolymer of N-acetylacrylamide with N-isopropyl acrylamide

1.0 g of N-acetyl acrylamide and 200 mg of N-isopropyl acrylamide weredissolved in 5 ml of ethanol in a three-necked flask. To the solutionwas then added 5 mg of AIBN. The mixture was then stirred at atemperature of 70° C. for 4 hours. The polymer thus precipitated waswashed with ethanol, and then thoroughly dried under reduced pressure toobtain 780 mg of a copolymer (weight-average molecular weight: about7,000).

25 mg of the copolymer thus obtained was then dissolved in 5 ml of a 15%aqueous solution of ethanol. The copolymer solution was then measuredfor upper critical solution temperature (UCST). As a result, it was 5°C. The same sample was then measured for lower critical solutiontemperature (LCST). As a result, it was 83° C.

The upper critical solution temperature (UCST) and the lower criticalsolution temperature (LCST) were determined as calculated in terms ofvisible light transmittance.

COMPARATIVE EXAMPLE 2-1 Synthesis and physical properties ofpoly-N-isopropyl acrylamide (PNIPAM)

In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamide and 5mg of AIBN were dissolved in ethylene glycol dimethyl ether, and thencharged into a flask where it was then stirred at a temperature of 75°C. for 3 hours. The reaction solution thus obtained was thenreprecipitated from a 10/1 mixture of cyclohexane and ethyl acetate toobtain 0.6 g of a white solid.

50 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was about 30° C.The aqueous solution was then allowed to stand at a temperature of 1° C.for 1 week. As a result, no polymers were observed precipitated. Thisdemonstrates that the polymer has no upper critical solution temperature(UCST).

In accordance with the second aspect of the present invention, athermo-responsive polymer having an upper critical solution temperature(UCST) and a lower critical solution temperature (LCST) can be obtained.The thermo-responsive polymer of the present invention is particularlyuseful for the separation, purification, fixing, calibration or controlof substances the working temperature of which can hardly bepredetermined (protein such as biological product, enzyme and antibody)or can be effectively used for chemovalve, etc.

EXAMPLE 3-2 Synthesis and physical properties of 1:1 copolymer ofN-acetyl acrylamide and N-acetyl methacrylamide

In an atmosphere of nitrogen gas, 1.1 g of N-acetyl acrylamide, 1.2 g ofN-acetyl methacrylamide and 5 mg of AIBN were dissolved in 10 ml ofdimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 200 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 2.0 g of a white solid. The white solid had a weight-averagemolecular weight of about 7,000.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 53° C.Further, the polymer solution showed a transition temperature range asvery sharp as 4° C.

Similarly, 25 mg of the same polymer was dissolved in a 0.01 N aqueoussolution of caustic soda. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, it was 66° C.

Similarly, 25 mg of the same polymer was dissolved in a 0.1 N aqueoussolution of caustic soda. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, the turbidpoint disappeared.

Similarly, 25 mg of the same polymer was dissolved in a 25% aqueoussolution of ethanol. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, the lower criticalsolution temperature (LCST) disappeared, and an upper critical solutiontemperature (UCST) was observed at a temperature of 50° C.

The polymer reversibly underwent dissolution and precipitation at thispoint many times. The transition temperature was measured as calculatedin terms of visible light transmittance.

EXAMPLE 3-3 Synthesis and physical properties of 1:4 copolymer ofN-acetyl acrylamide and N-acetyl methacrylamide

In an atmosphere of nitrogen gas, 1.1 g of N-acetyl acrylamide, 4.8 g ofN-acetyl methacrylamide and 5 mg of AIBN were dissolved in 20 ml ofdimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 500 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 5.5 g of a white solid.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 15° C.Further, the polymer solution showed a transition temperature range asvery sharp as 8° C.

Similarly, 25 mg of the same polymer was dissolved in a 0.1 N aqueoussolution of caustic soda and a 0.01 N aqueous solution of caustic soda,respectively. The polymer solutions were then measured for lowercritical solution temperature (LCST). As a result, the turbid pointdisappeared.

The polymer reversibly underwent dissolution and precipitation at thispoint many times. The transition temperature was measured as calculatedin terms of visible light transmittance.

EXAMPLE 3-4 Synthesis and physical properties of 2:3 copolymer ofN-acetyl acrylamide and N-acetyl methacrylamide

In an atmosphere of nitrogen gas, 2.2 g of N-acetyl acrylamide, 2.4 g ofN-acetyl methacrylamide and 5 mg of AIBN were dissolved in 20 ml ofdimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 400 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 4.2 g of a white solid.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 23° C.Further, the polymer solution showed a transition temperature range asvery sharp as 5° C.

Similarly, 25 mg of the same polymer was dissolved in 5 ml of a 0.01 Nhydrochloric acid. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 25° C.Further, the polymer solution showed a transition temperature range asvery sharp as 4° C.

Similarly, 25 mg of the same polymer was dissolved in a 0.01 N aqueoussolution of caustic soda. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, it was 68° C.

Similarly, 25 mg of the same polymer was dissolved in a 25% aqueoussolution of caustic soda. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, the turbidpoint disappeared.

Similarly, 25 mg of the same polymer was dissolved in a 30% aqueoussolution of ethanol. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, the lower criticalsolution temperature (LCST) disappeared, and an upper critical solutiontemperature (UCST) was observed at a temperature of 63° C.

The polymer reversibly underwent dissolution and precipitation at thispoint many times. The transition temperature was measured as calculatedin terms of visible light transmittance.

COMPARATIVE EXAMPLE 3-1 Synthesis and physical properties ofpoly-N-isopropyl acrylamide (PNIPAM)

In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamide and 5mg of AIBN were dissolved in 5 ml of ethylene glycol dimethyl ether, andthen charged in a flask where it was then stirred at a temperature of75° C. for 3 hours. The resulting reaction solution was thenrecrystallized from a 10/1 mixture of cyclohexane and ethyl acetate toobtain 0.6 g of a white solid.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water, a 0.01 N hydrochloric acid, a 0.01 N aqueous solutionof caustic soda and a 0.1 N aqueous solution of caustic soda. Thepolymer solution was then measured for lower critical solutiontemperature (LCST). As a result, these polymer solutions showed littleor no dependence of the LCST on pH and a lower critical solutiontemperature (LCST) of about 30° C.

25 mg of the polymer was dissolved in a 20% aqueous solution of ethanol.The polymer solution thus obtained was then measured for turbid point.As a result, the polymer solution showed a lower critical solutiontemperature (LCST) at a temperature of 19° C. but showed no uppercritical solution temperature (UCST).

In accordance with the third aspect of the present invention, astimuli-responsive polymer which can switch between lower criticalsolution temperature (LCST) and upper critical solution temperature(UCST) or allow individual occurrence of reversible dissolution andprecipitation in a single compound depending on the hydrogen ionconcentration can be obtained. The stimuli-responsive polymer of thepresent invention is particularly useful for the separation,purification, fixing, calibration or control of substances the workingtemperature of which can hardly be predetermined (protein such asbiological product, enzyme and antibody) or can be effectively used forchemovalve, etc.

EXAMPLE 4-1 Synthesis and physical properties of 1:12 copolymer ofN-acetyl acrylamide and acrylamide

In an atmosphere of nitrogen gas, 100 mg of N-acetyl acrylamide, 1.2 gof acrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide, and then charged in a flask where it was then stirred at atemperature of 75° C. for 3 hours. 200 ml of ethanol and a stirrer werethen put in a beaker. To ethanol was then gradually added dropwise theabove described reaction solution with vigorous stirring by a magneticstirrer. The mixture was then stirred for 2 hours. The resultingprecipitate was withdrawn by filtration, thoroughly washed with ethanol,and then dried at room temperature under reduced pressure to obtain 900mg of a white solid.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for uppercritical solution temperature (UCST). As a result, it was 24° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-2 Synthesis and physical properties of 1:11 copolymer ofN-acetyl acrylamide and acrylamide

The procedure of polymerization reaction and purification of Example 1was followed except that 100 mg of N-acetyl acrylamide, 1.1 g ofacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 850 mg of awhite solid was obtained.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 12° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-3 Synthesis and physical properties of 1:9 copolymer ofN-acetyl acrylamide and acrylamide

The procedure of polymerization reaction and purification of Example 1was followed except that 100 mg of N-acetyl acrylamide, 900 mg ofacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 820 mg of awhite solid was obtained.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 4° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-4 Synthesis and physical properties of 1:12 copolymer ofN-acetyl acrylamide and methacrylamide

The procedure of polymerization reaction and purification of Example 1was followed except that 100 mg of N-acetyl acrylamide, 1.2 g ofmethacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 21° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-5 Synthesis and physical properties of 1:11 copolymer ofN-acetyl acrylamide and methacrylamide

The procedure of polymerization reaction and purification of Example 1was followed except that 100 mg of N-acetyl acrylamide, 1.1 g ofmethacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 45° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-6 Synthesis and physical properties of 1:10 copolymer ofN-acetyl acrylamide and methacrylamide

The procedure of polymerization reaction and purification of Example 1was followed except that 100 mg of N-acetyl acrylamide, 1.0 g ofmethacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 55° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

The measurement of the transition temperature of the samples of Examples1 to 6 were all effected as calculated in terms of visible lighttransmittance. Further, all these copolymers repeatedly underwentreversible dissolution and precipitation even in physiological saline,though showing slightly different upper critical solution temperatures(UCST).

EXAMPLE 4-7 Synthesis and separating properties of 1:8 copolymer ofN-acetyl methacrylamide and methacrylamide having bithion fixed therein

100 mg of an acrylic acid ester represented by the following generalformula d, 100 mg of N-acetyl acrylamide, 1.2 g of methacrylamide and 5mg of AIBN were dissolved in 10 ml of dimethyl sulfoxide, and thencharged in a flask where it was then subjected to polymerizationreaction and purification under the same conditions as mentioned aboveto obtain 760 mg of a white solid.

35° C. of the polymer thus obtained was dissolved in an aqueous solutioncontaining 10 mg of crude avidin (purity: 85% as determined byhigh-performance liquid chromatography), and then cooled to 5° C. Theresulting precipitate was withdrawn by filtration, washed with 5° C. 10%brine, and then filtered. The resulting filtrate was dialyzed through adialysis tube, and then lyophilized to obtain 2 mg of avidin. The purityof avidin thus obtained was analyzed by high-performance liquidchromatography. As a result, it was 99.8%.

EXAMPLE 4-8 Chemical-Releasing Capsule

100 mg of N-acetyl acrylamide, 1.1 g of methacrylamide, 30 mg of N,N′-methylene bisacrylamide and 5 mg of ammonium persulfate weredissolved in 10 ml of distilled water. The solution was then reacted ata temperature of 10° C. to prepare a gel.

The gel thus prepared was then allowed to swell in 42° C. physiologicalsaline. To the gel was then added an aqueous solution of taxol. In thismanner, taxol was allowed to permeate into the gel overnight.Thereafter, the system was cooled to a temperature of 10° C. The gel waswithdrawn, thoroughly washed with a low temperature saline, and thendipped in 38° C. physiological saline for 1 hour. The saline was thenanalyzed by high-performance liquid chromatography. As a result, taxolwas confirmed released. Further, the gel was dipped in 10° C.physiological saline. The saline was then analyzed by high-performanceliquid chromatography. The release of the chemical was suspended. Thus,no taxol was identified.

In accordance with the fourth aspect of the present invention, athermo-responsive polymer which exhibits an upper critical solutiontemperature (UCST) in an aqueous solution, particularly in physiologicalsaline, can be obtained. The thermo-responsive polymer of the presentinvention is particularly useful for the separation, purification,fixing, calibration or control of substances the working temperature ofwhich can hardly be predetermined (protein such as biological product,enzyme and antibody) or can be effectively used for chemovalve, etc.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A pH-responsive polymer utilizing keto-enol tautomerism whichcomprises a polymerizable monomer component which is trifluoroethylpoly-N-methacryoylcarbamate.